Steroids as agonists for fxr

ABSTRACT

The invention relates to compounds of formula (I): 
     
       
         
         
             
             
         
       
     
     wherein R is ethyl and pharmaceutically acceptable salts, solvates or amino acid conjugates thereof. The compounds of formula (I) are useful as FXR agonists.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/602,307, filed Nov. 21, 2006, which is a continuation of U.S. patentapplication Ser. No. 10/471,549, filed Sep. 11, 2003, now U.S. Pat. No.7,138,390, which claims priority under 35 U.S.C. Section 371 to PCTPatent Application No. PCT/EP02/01832, filed Feb. 21, 2002, which claimspriority to U.S. Patent Application No. 60/274,959, filed Mar. 12, 2001,the disclosures of each of which are incorporated by reference herein intheir entirety.

BACKGROUND OF THE INVENTION

The present invention relates to Farnesoid X receptors (FXR). Moreparticularly, the present invention relates to compounds useful asagonists for FXR, pharmaceutical formulations comprising such compounds,and therapeutic use of the same.

Farnesoid X Receptor (FXR) is an orphan nuclear receptor initiallyidentified from a rat liver eDNA library (B M. Forman, et al., Cell81:687-693 (1995)) that is most closely related to the insect ecdysonereceptor. FXR is a member of the nuclear receptor family ofligand-activated transcription factors that includes receptors for thesteroid, retinoid, and thyroid hormones (D J. Mangelsdorf, et al., Cell83:841-850 (1995)). Northern and in situ analysis show that FXR is mostabundantly expressed in the liver, intestine, kidney, and adrenal (B M.Forman, et al., Cell 81:687-693 (1995) and W. Seol, et al., Mol.Endocrinnol. 9:72-85 (1995)). FXR binds to DNA as a heterodimer with the9-cis retinoic acid receptor (RXR). The FXR/R heterodimer preferentiallybinds to response elements composed of two nuclear receptor half sitesof the consensus AG(G/T)TCA organized as an inverted repeat andseparated by a single nucleotide (IR-1 motif) (B M. Forman, et al., Cell81:687-693 (1995)). An early report showed that rat FXR is activated bymicromolar concentrations of farnesoids such as farnesol and juvenilehormone (B M. Forman, et al., Cell 81:687-693 (1995)). However, thesecompounds failed to activate the mouse and human FXR, leaving the natureof the endogenous FXR ligand in doubt. Several naturally-occurring bileacids bind to and activate FXR at physiological concentrations (PCT WO00/37077, published 29 Jun. 2000)). As discussed therein, the bile acidsthat serve as FXR ligands include chenodeoxycholic acid (CDCA),deoxycholic acid (DCA), lithocholic acid (LCA), and the taurine andglycine conjugates of these bile acids.

Bile acids are cholesterol metabolites that are formed in the liver andsecreted into the duodenum of the intestine, where they have importantroles in the solubilization and absorption of dietary lipids andvitamins. Most bile acids (˜95%) are subsequently reabsorbed in theileum and returned to the liver via the enterohepatic circulatorysystem. The conversion of cholesterol to bile acids in the liver isunder feedback regulation: Bile acids down-regulate the transcription ofcytochrome P450 7a (CYP7a), which encodes the enzyme that catalyzes therate limiting step in bile acid biosynthesis. There are data to suggestthat FXR is involved in the repression of CYP7a expression by bileacids, although the precise mechanism remains unclear (D W. Russell,Cell 97:539-542 (1999)). In the ileum, bile acids induce the expressionof the intestinal bile acid binding protein (IBABP), a cytoplasmicprotein which binds bile acids with high affinity and may be involved intheir cellular uptake and trafficking. Two groups have now demonstratedthat bile acids mediate their effects on IBABP expression throughactivation of FXR, which binds to an IR-1 type response element that isconserved in the human, rat, and mouse IBABP gene promoters (14;17).Thus FXR is involved in both the stimulation (IBABP) and the repression(CYP7a) of target genes involved in bile acid and cholesterolhomeostasis.

European Patent No. 0 312 867, published 5 May 1992 to Giuliana S. p. A.describes 6-methyl derivatives of natural biliary acids such asursodeoxycholic acid, ursocholic acid, chenodeoxycholic acid and cholicacid.

BRIEF SUMMARY OF THE INVENTION

According to a first aspect, the present invention provides compounds offormula I:

wherein R is ethyl, and pharmaceutically acceptable salts, solvates oramino acid conjugates thereof In one preferred embodiment, the compoundof formula (I) is in the form of the glycine or taurine conjugate.

In another aspect, the present invention provides3α,7α-dihydroxy-6α-ethyl-1,5β-cholan-24-oic acid and pharmaceuticallyacceptable salts, solvates or amino acid conjugates thereof.

In another aspect, the present invention provides compounds which areFXR agonists.

In another aspect, the present invention provides a pharmaceuticalformulation comprising a compound of formula (I) and a pharmaceuticallyacceptable carrier or diluent.

In another aspect, the present invention provides a method for theprevention or treatment of an FXR mediated disease or condition. Themethod comprises administering a therapeutically effective amount of acompound of formula (I). The present invention also provides the use ofa compound of formula (I) for the preparation of a medicament for theprevention or treatment of an FXR mediated disease or condition.

In another aspect, the present invention provides a method for theprevention or treatment of cardiovascular disease. The method comprisesadministering a therapeutically effective amount of a compound offormula (I). The present invention also provides the use of a compoundaccording to claim 1 for the preparation of a medicament for theprevention or treatment of cardiovascular disease. In one embodiment,the cardiovascular disease is atherosclerosis.

In another aspect, the present invention provides a method forincreasing HDL cholesterol. The method comprises administering atherapeutically effective amount of a compound of formula (I). Thepresent invention also provides the use of a compound according to claimI for the preparation of a medicament for increasing HDL-cholesterol.

In another aspect, the present invention provides a method for loweringtriglycerides. The method comprises administering a therapeuticallyeffective amount of a compound of formula (I). The present inventionalso provides the use of a compound according to claim 1 for thepreparation of a medicament for lowering triglycerides.

In another aspect, the present invention provides a method for theprevention or treatment of cholestatic liver disease. The methodcomprises administering a therapeutically effective amount of a compoundof formula (I). The present invention also provides the use of acompound according to claim 1 for the preparation of a medicament forthe prevention or treatment of cholestatic liver diseases.

In another aspect, the present invention provides a radiolabeledcompound of formula (I). In one embodiment, the compound of formula (I)is tritiated.

In another aspect, the present invention provides a process forpreparing a compound of formula (I) and pharmaceutically acceptablesalts, solvates or amino acid conjugates thereof. The process comprisesthe steps of:

a) reacting 3α-hydroxy-7-keto-5β-cholan-24-oic acid with3,4-dihydropyrane to prepare3α-tetrahydropyranyloxy-7-keto-5β-cholan-24-oic acid;

b) reacting 3α-tetrahydropyranyloxy-7-keto-5β-cholan-24-oic acid with analkyl bromide of the formula R—Br where R is ethyl to prepare a compoundof formula (II)

wherein R is ethyl;

c) reacting the compound of formula (II) with sodium borohydride toprepare a compound of formula (III)

d) reacting the compound of formula (III) with sodium hydroxide toprepare the compound of formula (I).

Further aspects of the present invention are described in the detaileddescription of the invention, examples, and claims which follow.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds of formula I:

wherein R is ethyl and pharmaceutically acceptable salts, solvates oramino acid conjugates thereof

Suitable pharmaceutically acceptable salts according to the presentinvention will be readily determined by one skilled in the art and willinclude, for example, basic salts such as metallic salts made fromaluminium, calcium, lithium, magnesium, potassium, sodium, and zinc ororganic salts made from N,N¹-dibenzylethylenediamine, chlorprocaine,choline, diethanolamine, ethylendiamine, meglumine (N-methylglucamine),and procaine. Such salts of the compounds of formula (I) may be preparedusing conventional techniques, from the compound of Formula (I) byreacting, for example, the appropriate base with the compound of Formula(I).

When used in medicine, the salts of a compound of formula (I) should bepharmaceutically acceptable, but pharmaceutically unacceptable salts mayconveniently be used to prepare the corresponding free base orpharmaceutically acceptable salts thereof.

As used herein, the term “solvate” is a crystal form containing thecompound of formula (I) or a pharmaceutically acceptable salt thereofand either a stoichiometric or a non-stoichiometric amount of a solvent.Solvents, by way of example, include water, methanol, ethanol, or aceticacid. Hereinafter, reference to a compound of formula (I) is to anyphysical form of that compound, unless a particular form, salt orsolvate thereof is specified.

As used herein, the term “amino acid conjugates” refers to conjugates ofthe compounds of formula (I) with any suitable amino acid. Preferably,such suitable amino acid conjugates of the compounds of formula (I) willhave the added advantage of enhanced integrity in bile or intestinalfluids. Suitable amino acids include but are not limited to glycine andtaurine. Thus, the present invention encompasses the glycine and taurineconjugates of any of the compounds of formula (I).

Preferred compounds of formula (I) include3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid and its pharmaceuticallyacceptable salts, solvates or amino acid conjugates thereof.

Hereinafter all references to “compounds of formula (I)” refer tocompounds of formula (I) as described above together with their andpharmaceutically acceptable salts, solvates or amino acid conjugatesthereof.

Preferably, the compounds of formula (I) are FXR agonists. As usedherein, the term “agonist” refers to compounds which achieve at least50% activation of FXR relative to CDCA, the appropriate positive controlin the assay methods described in PCT Publication No. WO 00/37077published 29 Jun. 2000 to Glaxo Group Limited, the subject matter ofwhich is incorporated herein by reference in its entirety. Morepreferably, the compounds of this invention achieve 100% activation ofFXR in the scintillation proximity assay or the HTRF assay as describedin PCT Publication No. WO 00/37077.

The compounds of the formula (I) are useful for a variety of medicinalpurposes. The compounds of formula (I) may be used in methods for theprevention or treatment of FXR mediated diseases and conditions. FXRmediated diseases or conditions include cardiovascular diseasesincluding atherosclerosis, arteriosclerosis, hypercholesteremia, andhyperlipidemia. In particular, the compounds of formula (I) are usefulin the treatment and prevention of cardiovascular disease includingatherosclerosis and hypercholesteremia. The compounds of formula (I) arealso useful for increasing HDL-cholesterol, and lowering triglycerides.

In addition, the compounds of the present invention are useful for theprevention and treatment of cholestatic liver diseases. The compounds ofthe present invention increase the flow of bile acid. Increased flow ofbile acids improves the flux of bile acids from the liver to theintestine. See, C. Sinal, Cell 102: 731-744 (2000). Essentially, FXRnull mice demonstrate that FXR plays a central role in bile acidhomeostasis, and is therefore critical to lipid homeostasis by virtue ofthe regulation of enzymes and transporters that are critical to lipidcatabolism and excretion. FXR therefore is an important target for thetreatment of a number of cholestatic liver diseases and other lipidrelated diseases and conditions.

The methods of the present invention are useful for the treatment ofmammals generally and particularly humans.

The methods of the present invention comprise the step of administeringa therapeutically effective amount of the compound of formula (I). Asused herein, the term “therapeutically effective amount” refers to anamount of the compound of formula (I) which is sufficient to achieve thestated effect. Accordingly, a therapeutically effective amount of acompound of formula (I) used in the method for the prevention ortreatment of FXR mediated diseases or conditions will be an amountsufficient to prevent or treat the FXR mediated disease or condition.Similarly, a therapeutically effective amount of a compound of formula(I) for use in the method for the prophylaxis or treatment ofcholestatic liver diseases or increasing bile flow will be an amountsufficient to increase bile flow to the intestine.

The amount of a compound of formula (I) or pharmaceutically acceptablesalt or solvate thereof which is required to achieve the desiredbiological effect will depend on a number of factors such as the use forwhich it is intended, the means of administration, and the recipient,and will be ultimately at the discretion of the attendant physician orveterinarian. In general, a typical daily dose for the treatment of FXRmediated diseases and conditions, for instance, may be expected to liein the range of from about 0.01 mg/kg to about 100 mg/kg. This dose maybe administered as a single unit dose or as several separate unit dosesor as a continuous infusion. Similar dosages would be applicable for thetreatment of other diseases, conditions and therapies including theprophylaxis and treatment of cholestatic liver diseases.

Thus in a further aspect the present invention provides pharmaceuticalcompositions comprising, as active ingredient, a compound of formula (I)or a pharmaceutically acceptable salt or solvate thereof, together withat least one pharmaceutical carrier or diluent. These pharmaceuticalcompositions may be used in the prophylaxis and treatment of theforegoing diseases or conditions and in cardiovascular therapies asmentioned above.

The carrier must be pharmaceutically acceptable and must be compatiblewith, i.e. not have a deleterious effect upon, the other ingredients inthe composition. The carrier may be a solid or liquid and is preferablyformulated as a unit dose formulation, for example, a tablet which maycontain from 0.05 to 95% by weight of the active ingredient. If desiredother physiologically active ingredients may also be incorporated in thepharmaceutical compositions of the invention.

Possible formulations include those suitable for oral, sublingual,buccal, parenteral (for example subcutaneous, intramuscular, orintravenous), rectal, topical including transdermal, intranasal andinhalation administration. Most suitable means of administration for aparticular patient will depend on the nature and severity of the diseaseor condition being treated or the nature of the therapy being used andon the nature of the active compound, but where possible, oraladministration is preferred for the prevention and treatment of FXRmediated diseases and conditions.

Formulations suitable for oral administration may be provided asdiscrete units, such as tablets, capsules, cachets, lozenges, eachcontaining a predetermined amount of the active compound; as powders orgranules; as solutions or suspensions in aqueous or non-aqueous liquids;or as oil-in-water or water-in-oil emulsions.

Formulations suitable for sublingual or buccal administration includelozenges comprising the active compound and, typically a flavoured base,such as sugar and acacia or tragacanth and pastilles comprising theactive compound in an inert base, such as gelatine and glycerine orsucrose acacia.

Formulations suitable for parenteral administration typically comprisesterile aqueous solutions containing a predetermined concentration ofthe active compound; the solution is preferably isotonic with the bloodof the intended recipient. Additional formulations suitable forparenteral administration include formulations containingphysiologically suitable co-solvents and/or complexing agents such assurfactants and cyclodextrins. Oil-in-water emulsions are also suitableformulations for parenteral formulations. Although such solutions arepreferably administered intravenously, they may also be administered bysubcutaneous or intramuscular injection.

Formulations suitable for rectal administration are preferably providedas unit-dose suppositories comprising the active ingredient in one ormore solid carriers forming the suppository base, for example, cocoabutter.

Formulations suitable for topical or intranasal application includeointments, creams, lotions, pastes, gels, sprays, aerosols and oils.Suitable carriers for such formulations include petroleum jelly,lanolin, polyethyleneglycols, alcohols, and combinations thereof.

Formulations of the invention may be prepared by any suitable method,typically by uniformly and intimately admixing the active compound withliquids or finely divided solid carriers or both, in the requiredproportions and then, if necessary, shaping the resulting mixture intothe desired shape.

For example a tablet may be prepared by compressing an intimate mixturecomprising a powder or granules of the active ingredient and one or moreoptional ingredients, such as a binder, lubricant, inert diluent, orsurface active dispersing agent, or by moulding an intimate mixture ofpowdered active ingredient and inert liquid diluent.

Suitable formulations for administration by inhalation include fineparticle dusts or mists which may be generated by means of various typesof metered dose pressurised aerosols nebulisers, or insufflators.

For pulmonary administration via the mouth, the particle size of thepowder or droplets is typically in the range 0.5-10 μm, preferably 1-5μm, to ensure delivery into the bronchial tree. For nasaladministration, a particle size in the range 10-500 μm is preferred toensure retention in the nasal cavity.

Metered dose inhalers are pressurised aerosol dispensers, typicallycontaining a suspension or solution formulation of the active ingredientin a liquefied propellant. During use, these devices discharge theformulation through a valve adapted to deliver a metered volume,typically from 10 to 150 μl, to produce a fine particle spray containingthe active ingredient. Suitable propellants include certainchlorofluorocarbon compounds, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.The formulation may additionally contain one or more co-solvents, forexample, ethanol surfactants, such as oleic acid or sorbitan trioleate,anti-oxidants and suitable flavouring agents.

Nebulisers are commercially available devices that transform solutionsor suspensions of the active ingredient into a therapeutic aerosol misteither by means of acceleration of a compressed gas typically air oroxygen, through a narrow venturi orifice, or by means of ultrasonicagitation. Suitable formulations for use in nebulisers consist of theactive ingredient in a liquid carrier and comprising up to 40% w/w ofthe formulation, preferably less than 20% w/w. The carrier is typicallywater or a dilute aqueous alcoholic solution, preferably made isotonicwith body fluids by the addition of, for example, sodium chloride.Optional additives include preservatives if the formulation is notprepared sterile, for example, methyl hydroxy-benzoate, anti-oxidants,flavouring agents, volatile oils, buffering agents and surfactants.

Suitable formulations for administration by insufflation include finelycomminuted powders which may be delivered by means of an insufflator ortaken into the nasal cavity in the manner of a snuff. In theinsufflator, the powder is contained in capsules or cartridges,typically made of gelatin or plastic, which are either pierced or openedin situ and the powder delivered by air drawn through the device uponinhalation or by means of a manually-operated pump. The powder employedin the insufflator consists either solely of the active ingredient or ofa powder blend comprising the active ingredient, a suitable powderdiluent, such as lactose, and an optional surfactant. The activeingredient typically comprises from 0.1 to 100 w/w of the formulation.

In addition to the ingredients specifically mentioned above, theformulations of the present invention may include other agents known tothose skilled in the art of pharmacy, having regard for the type offormulation in issue. For example, formulations suitable for oraladministration may include flavouring agents and formulations suitablefor intranasal administration may include perfumes.

Therefore, according to a further aspect of the present invention, thereis provided the use of a compound of formula (I) in the preparation of amedicament for the prevention or treatment of FXR mediated diseases orconditions.

Compounds of the invention can be made according to any suitable methodof organic chemistry. According to one method, compounds of formula (I)are prepared using the synthesis process as depicted in Scheme 1:

wherein R is ethyl.

Generally, the compounds of the present invention can be prepared by theprocess comprising a) reacting 3α-hydroxy-7-keto-5β-cholan-24-oic acidwith 3,4-dihydropyrane to prepare3α-tetrahydropyranyloxy-7-keto-5β-cholan-24-oic acid; b) reacting3α-tetrahydropyranyloxy-7-keto-5β-cholan-24-oic acid with an alkylbromide of the formula R—Br where R is ethyl to prepare a compound offormula (II); c) reacting the compound of formula (II) with sodiumborohydride to prepare a compound of formula (III); d) reacting thecompound of formula (III) with sodium hydroxide to prepare the compoundof formula (I).

More particularly, the compounds of formula (I) are convenientlyprepared by reacting the compounds of formula (III) with sodiumhydroxide in a suitable solvent at ambient temperature. Suitablesolvents include lower alcohols, such as ethanol. The reaction mixturemay optionally be acidified with an appropriate acid such ashydrochloric acid.

The compounds of formula (III) are conveniently prepared by reactingcompounds of formula (II) with sodium borohydride in a suitable solventat ambient temperature. Suitable solvents include lower alcohols such asethanol.

The compounds of formula (II) are conveniently prepared by reacting3α-tetrahydropyranyloxy-7-keto-5β-cholan-24-oic acid with an alkylbromide of the formula R—Br where R is ethyl in a suitable solvent andin the presence of n-Butyl lithium and HMPA in diisopropylamine. Polarsolvents such as tetrahydrofuran are useful for conducting the reaction.Preferably, the reaction is carried out at cold temperatures such asabout −70 to −80° C.

3α-Tetrahydropyranyloxy-7-keto-5β-cholan-24-oi acid can conveniently beprepared from 3α-hydroxy-7-keto-5β-cholan-24-oic acid by reacting with3,4-dihydropyrane in p-toluenesulfonic acid.

Pharmaceutically acceptable salts, solvates and amino acid conjugates ofthe compounds of formula (I) can be prepared from the free base usingmethods known to those skilled in the art.

The present invention also provides radiolabeled compounds of formula(I). Radiolabeled compounds of formula (I) can be prepared usingconventional techniques. For example, radiolabeled compounds of formula(I) can be prepared by reacting the compound of formula (I) with tritiumgas in the presence of an appropriate catalyst to produce radiolabeledcompounds of formula (I). In one preferred embodiment, the compounds offormula (I) are tritiated.

The radiolabeled compounds of formula (I) are useful in assays for theidentification of compounds which interact with FXR such as thosedescribed in PCT Publication No. WO 00/37077 already incorporatedherein.

The following examples are intended for illustration only and are notintended to limit the scope of the invention in any way, the presentinvention being defined by the claims.

Example Synthesis of 3α7α-Dihydroxy-6α-ethyl-5β-cholan-24-oic Acid(6αEt-CDCA) 3α-Tetrahydropyranyloxy-7-keto-5β-cholan-24-oic acid

p-Toluensulfonic acid (6.0 g, 3.2 mmol) and 3,4-dihydro-2H-pyrane (4.6g, 54 mmol) were added to a solution of3α-hydroxy-7-keto-5β-cholan-24-oic acid (1) (6.0 g, 14.4 mmol) in 120 mlof dioxane. The reaction mixture was stirred at room temperature for 15min and then was treated with methanol saturated with ammonia until itreached pH of about 8-9. The solvents were removed under vacuum and theresidue was extracted with chloroform (200 ml) and washed with asaturated NaHCO₃ solution (2×50 ml). After drying over anhydrous Na₂SO₄and evaporation under vacuum, the residue was purified by silica gelchromatography. Elution by CHCl₃:MeOH (90:10) yielded 5.4 g (10.4 mmol,74% yield) of compound 2 as a white solid (mp: 157-159° C.).

¹H-NMR (CDCl₃) δ: 0.58 (s, 3H, CH₃-18); 0.88 (d, J=6.1 Hz, 3H, CH₃-21);1.14 (s, 3H, CH₃-19); 3.3-3.7 (m, 3H, pyr); 3.75-3.95 (m, 1H, pyr);4.64-4.71 (m, 1H, CH-3).

Ethyl 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oate

n-Butyl lithium (21.1 ml, 1.6M solution in hexane) and HMPA (4.3 ml)were added dropwise at −78° C. to a solution of diisopropylamine (4.1ml, 33.7 mmol) in 250 ml of dry THF. The system was held at −78° C. foran additional 30 min and then,3α-tetrahydropyranyloxy-7-keto-5β-cholan-24-oic acid (2) (5 g, 10.5mmol) dissolved in 50 ml of dry THF was cooled to −78° C. and addeddropwise to the mixture. After 20 minutes ethyl bromide (7.8 ml, 105mmol) dissolved in THF (20 ml) was slowly added and the mixture wasallowed to come to room temperature overnight. The solvents were removedunder vacuum, acidified by 10% HCl and extracted with ethyl acetate(5×200 ml), and washed with a saturated NaCl solution (1×200 ml). Afterdrying over anhydrous Na₂SO₄ and evaporation under vacuum, the cruderesidue was refluxed with a solution of 2N HCl in EtOH (50 ml) for 12hours. The residue was evaporated under vacuum and extracted with ethylacetate (300 ml), washed with a saturated NaHCO₃ solution (2×100 ml),dried with Na₂SO₄ and evaporated under vacuum. The residue was purifiedby silica gel chromatography; elution by light petroleum:ethyl acetate(70:30) yielded 0.57 g (1.27 mmol, 12% yield) of ethyl3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oate (3) as an amorphous solid.

¹H-NMR (CDCl₃) δ: 0.50 (s, 3H, CH₃-18); 0.69 (t, J=7.3 Hz, 31-1, CH₂—CH₃); 0.82 (d, J=6.2 Hz, 3H, CH₃-21); 1.06-1.18 (m, 8H, CO₂ CH₂ CH+CH₂—CH₃+CH₃-19); 3.36-3.42 (m, 1H, CH—OH), 4.01 (q, J=7.2, Hz 2H, CO₂ CH₂CH₃).

Ethyl 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oate

Ethyl 3α-hydroxy-6α-ethyl-7-keto-5β-cholan-24-oate (3) (0.185 g, 0.4mmol) was dissolved in 30 ml of 96% EtOH and treated with NaBH₄ (30 mg,0.8 mmol). The mixture was stirred at room temperature for 2 hours.Water (10 ml) was then added and the mixture was partially concentratedunder vacuum and extracted with ethyl acetate (3×20 ml). The combinedorganic fractions were washed with a saturated NaCl solution (1×50 ml),dried with Na₂SO₄ and evaporated under vacuum. To give ethyl3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oate (4) (0.15 g, 0.33 mmol, 81%yield) as white solid (mp: 55-57° C.).

¹H-NMR (CDCl₃) δ: 0.62 (s, 3H, CH₃-18); 0.84-0.92 (m, 9H, CH₂—CH₃+CH₃-19+CH₃-21); 1.22 (t, J=7.2 Hz, 3H, CO₂ CH₂ CH ₃); 3.30-3.47 (m,1H, CH-3), 3.66 (brs, 1H, CH-7); 4.08 (q, J=7.2, Hz 2H, CO₂ CH ₂CH₃).

3α,7α-Dihydroxy-6α-ethyl-5β-cholan-24-oic acid

Ethyl 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oate (4) (0.10 g, 0.22 mmol)was dissolved in 15 ml of 96% EtOH and added to 10% NaOH in 96% EtOH (2ml, 5 mmol). The mixture was refluxed for 4 hours. The mixture wasacidified with 3N HCl and extracted with ethyl acetate (3×20 ml). Thecombined organic fractions were washed with a saturated NaCl solution(1×50 ml), dried with Na₂SO₄ and evaporated under vacuum. The residuewas chromatographed on silica gel column; elution by CHCl₃:MeOH (95:5)yielded 3α,7α-dihydroxy-6α-methyl-5β-cholan-24-oic acid (6) (0.04 g,0.095 mmol, 43% yield).

¹H-NMR (CDCl₃) δ: 0.67 (s, 3H, CH₃-18); 0.90-0.96 (m, 9H, CH₂—CH₃+CH₃-19+CH₃-21); 2.22-2.46 (2m, 2H, CH₂-23); 3.39-3.47 (m, 1H, CH-3),3.72 (brs, 1H, CH-7).

¹³C-NMR (CDCl₃) δ: 11.65, CH₂ CH ₃-6; 11.80, C-18; 18.25, C-21, 20.76,C-11; 22.23, CH ₂CH₃-6; 23.14, C-19; 23.69, C-15; 28.17,C-16; 30.53,C-2; 30.81, C-22; 30.95, C-23; 33.23, C-9; 33.90, C-10; 35.38, C-20;35.52, C-1; 35.70, C-4; 39.60, C-12; 40.03, C-5; 41.19, C-6; 42.77,C-13; 45.19, C-8; 50.49, C-14; 55.80, C-17; 70.97, C-7; 72.38, C-3;179.19, C-24.

1. A method for the prevention or treatment of cholestatic liver diseasecomprising administering to a mammal a therapeutically effective amountof a compound of formula I:

or a pharmaceutically acceptable salt, solvent, or amino acid conjugatesthereof, wherein R is ethyl.
 2. The method according to claim 1, whereinthe compound is the glycine conjugate of a compound of formula I.
 3. Themethod according to claim 1, wherein the compound is the taurineconjugate of a compound of formula I.
 4. The method according to claim1, wherein the compound is the pharmaceutically acceptable salt of acompound of formula I.
 5. The method according to claim 1, wherein themammal is a human.